Membrane Protein Solid State NMR: PISEMA Development and the M2 Tetramer Structure

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Membrane proteins represent approximately 30% of all genomes yet characterized. Today, less than 40 unique membrane protein structures are in the Protein Data Bank. Membrane proteins carry out numerous critical functions for cells and they are vastly different from their water-soluble counterparts through: their amino acid composition, the forces that stabilize their structure, their internal dynamics, etc. Sold state NMR is a demonstrated technology for achieving 3D high resolution structure in a lipid bilayer environment. This technology uniquely utilizes fully hydrated lipid bilayers for solvating protein samples. This work will further develop the primary solid state NMR experiment used for the structural characterization, PISEMA. Several modifications including the use of ramped pulses during the Lee-Goldburg sequence suggests significant gains in making PISEMA a more robust experiment. Advanced NMR probes with balanced RF circuits in high field magnets available at the NHMFL will be a great help in achieving this goal. The analysis of PISEMA spectra of alpha-helical proteins has led to cover stories in both the Journal of Magnetic Resonance and Protein Science. The characterization of solid state NMR data leads directly to a description of the tilt and rotational orientation of the helix within the lipid bilayer. Here, this analysis will be advanced using mathematical expressions to show how degeneracies in the structural solutions are resolved, by showing that unique spectral patterns are predicted for alpha, 310, and pi helices, and by characterizing the more complex spectral patterns for helices in the plane of the lipid bilayer. Moreover, it is the aim of this project to solve the complete backbone structure and assembly of the 44 kDa tetrameric M2 proton channel in hydrated lipid bilayers above the phase transition of the lipids. In this project Dr. Cross and his colleagues will develop a unique approach for determining the three dimensional structure of membrane proteins in their native environment - the lipid membrane that surrounds each cell. These proteins control everything that enters and leaves the cells, and they are responsible for accepting and sending information between cells. 30% or more of the proteins of a given genome are membrane proteins and yet technologies are very limited for their structural characterization. With a unique team of chemists, mathematicians and biophysicists, Dr. Cross brings together the necessary skills for the development of a Nuclear Magnetic Resonance methodology that has now been demonstrated to achieve unique structures of membrane proteins. Dr. Cross and his coworkers are refining the technology and enhancing its sensitivity for greater applications. This research will have a broad impact in both the fields of membrane protein biophysics and structural biology and students in chemical, biological and mathematical disciplines will be trained in a proven interdisciplinary environment.